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Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

ABSTRACT: Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.

SUBMITTER: Wise JG

PROVIDER: S-EPMC3383123 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

Wise John G JG

Biochemistry 20120612 25

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to ...[more]

PMID: 22647192

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Project description:IntroductionHyperprolactinaemia, a common adverse effect of antipsychotic drugs, is primarily linked to blockade of dopamine D2 receptors in the pituitary gland. Certain antipsychotic drugs, such as, for example risperidone and paliperidone, are more likely to induce hyperprolactinaemia compared to others. This effect is probably caused by a relatively high blood/brain concentration ratio, a consequence of being a substrate of P-glycoprotein. Genetic variants of P-glycoprotein with changed functional activity might influence the potential of risperidone and paliperidone to cause hyperprolactinaemia as the altered blood/brain concentration ratio would lead to a reduced therapeutic drug level within essential brain areas making dose adaptations necessary. This increases exposure of dopamine D2 receptors within the pituitary gland.AimsTo investigate possible associations between MDR1/ABCB1 gene polymorphisms and antipsychotic drug-induced hyperprolactinaemia in Russian patients with schizophrenia and to determine possible differences between risperidone/paliperidone and other antipsychotics.MethodsIn total, 446 patients with schizophrenia were included from 3 psychiatric hospitals in Siberia. Blood samples were obtained in a cross-sectional study design for DNA extraction and prolactin measurement. Associations between hyperprolactinaemia and 8 MDR1/ABCB1 gene-polymorphisms were assessed using logistic regression analysis accounting for covariates. The analysis was repeated in a patient subgroup using risperidone or paliperidone.ResultsWe did not observe an association between any of the 8 single nucleotide polymorphisms and the prevalence of antipsychotic-induced hyperprolactinaemia in the total patient population. However, in the risperidone/paliperidone subgroup, the single nucleotide polymorphism rs2032582 (G2677T) was found to be negatively associated with risperidone/paliperidone-induced hyperprolactinaemia.ConclusionThis study revealed a significant association between the ABCB1 gene polymorphism rs2032582 (G2677T) and risperidone/paliperidone-induced hyperprolactinaemia.

Project description:BackgroundRaltegravir (Isentress®)(RALT) has demonstrated excellent efficacy in both treatment-experienced and naïve patients with HIV-1 infection, and is the first strand transfer integrase inhibitor to be approved for use in HIV infected adults worldwide. Since the in vivo efficacy of this class of antiviral drugs depends on their access to intracellular sites where HIV-1 replicates, we analyzed the biological effects induced by RALT on human MDR cell systems expressing multidrug transporter MDR1-P-glycoprotein (MDR1-Pgp).MethodsOur study about RALT was performed by using a set of consolidated methodologies suitable for evaluating the MDR1-Pgp substrate nature of chemical and biological agents, namely: i) assay of drug efflux function; ii) analysis of MDR reversing capability by using cell proliferation assays; iii) monoclonal antibody UIC2 (mAb) shift test, as a sensitive assay to analyze conformational transition associated with MDR1-Pgp function; and iv) induction of MDR1-Pgp expression in MDR cell variant subjected to RALT exposure.ResultsFunctional assays demonstrated that the presence of RALT does not remarkably interfere with the efflux mechanism of CEM-VBL100 and HL60 MDR cells. Accordingly, cell proliferation assays clearly indicated that RALT does not revert MDR phenotype in human MDR1-Pgp expressing cells. Furthermore, exposure of CEM-VBL10 cells to RALT does not induce MDR1-Pgp functional conformation intercepted by monoclonal antibody (mAb) UIC2 binding; nor does exposure to RALT increase the expression of this drug transporter in MDR1-Pgp expressing cells.ConclusionsNo evidence of RALT interaction with human MDR1-Pgp was observed in the in vitro MDR cell systems used in the present investigation, this incorporating all sets of studies recommended by the FDA guidelines. Taken in aggregate, these data suggest that RALT may express its curative potential in all sites were HIV-1 penetrates, including the MDR1-Pgp protected blood/tissue barrier. Moreover RALT, evading MDR1-Pgp drug efflux function, would not interfere with pharmacokinetic profiles of co-administered MDR1-Pgp substrate antiretroviral drugs.

Project description:P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

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Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

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Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites.

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